Contribution of efflux and mutations in fluoroquinolone susceptibility in MDR enterobacterial isolates: a quantitative and molecular study

Abstract

Objectives The emergence of MDR strains is a public health problem in the management of associated infections. Several resistance mechanisms are present, and antibiotic efflux is often found at the same time as enzyme resistance and/or target mutations. However, in the laboratory routinely, only the latter two are identified and the prevalence of antibiotic expulsion is underestimated, causing a misinterpretation of the bacterial resistance phenotype. The development of a diagnostic system to quantify the efflux routinely would thus improve the management of patients. Methods A quantitative technique based on detection of clinically used fluoroquinolones was investigated in Enterobacteriaceae clinical strains with a high or basal efflux activity. The detail of efflux involvement was studied from MIC determination and antibiotic accumulation inside bacteria. WGS was carried out on selected strains to determine the genetic background associated with efflux expression. Results Only 1 Klebsiella pneumoniae isolate exhibited a lack of efflux whereas 13 isolates had a basal efflux and 8 presented efflux pump overexpression. The antibiotic accumulation evidenced the efficacy of the efflux mechanism in strains, and the contribution of dynamic expulsion versus target mutations in fluoroquinolone susceptibility. Conclusions We confirmed that phenylalanine arginine β-naphthylamide is not a reliable marker of efflux due to the affinity of the AcrB efflux pump for different substrates. We have developed an accumulation test that can be used efficiently on clinical isolates collected by the biological laboratory. The experimental conditions and protocols ensure a robust assay that with improvements in practice, expertise and equipment could be transferred to the hospital laboratory to diagnose the contribution of efflux in Gram-negative bacteria.

RND Efflux Pump Induction: A Crucial Network Unveiling Adaptive Antibiotic Resistance Mechanisms of Gram-Negative Bacteria

“…Additionally, efforts should be directed towards exploring alternative strategies to target efflux pumps, either through the design of efflux pump inhibitors (EPIs) [ 126 ] or through the development of new antimicrobial agents that are less susceptible to efflux-mediated resistance [ 25 ]. Finally, the diagnosis of infection by bacteria overexpressing an efflux system needs to be developed and improved as a routine in hospitals and the community [ 9 ]. Although antibacterial resistance arises through various mechanisms, the increased active efflux of antibiotics is particularly significant.…”

“…Innate resistance refers to the natural resistance of bacterial species to specific antibiotics, as seen in Escherichia coli , with the intrinsic expression of AmpC, and of the AcrAB-TolC multi-drug efflux system [ 8 ]. Acquired resistance enables strains to enhance their resistance levels through mutations [ 9 ] or acquisition of genetic material from other bacteria [ 10 ]. Moreover, adaptive or induced resistance involves the occasional or excessive activation of previously described mechanisms in response to stress or resistance-inducing molecules.…”

Molecular determinant deciphering of MIC-guided RND efflux substrates in E. coli

“…Since E. coli is the most representative AMR pathogen and is frequently exposed to widely used antibiotics (Antimicrobial Resistance Collaborators, 2022), we studied the following effluxresistant strains from literature MIC data, comparing them within a single antibiotic family (Figure 1A): 1) marR mutants: AG102 (point mutations correlated with the loss of marR repressor function by inhibiting marO promoter binding, leading to marA overexpression) (Cohen et al, 1988;Alekshun and Levy, 1997;Randall and Woodward, 2002;Whalen et al, 2015;Yilmaz et al, 2015;Vergalli et al, 2020;Ferrand et al, 2023), AG102B (Okusu et al, 1996;Aires and Nikaido, 2005) and 3-AG100 (Bohnert et al, 2008;Wehmeier et al, 2009;Bohnert et al, 2016;Schuster et al, 2016); 2) marA mutants: AG112 and CH164 (marO deletion mutation leading to an affinity loss with marR repressor and therefore marA overexpression) (Oethinger et al, 2000;Chollet et al, 2004;Nicoloff et al, 2006). Additionally, we selected some recombined strains with plasmids: HN1157/pHSGoxa7 with acrR deletion and penicillinase OXA-7 gene plasmid (operon acrR and acrAB transcription repressor) (Lim and Nikaido, 2010), GC7368/ pCLL3431, an AG100A E. coli strain with cloned AcrAB gene leading to efflux pump overexpression (Visalli et al, 2003), JM101/pMAQ43, an MDR-derived strain with ramA (acrA overexpression in absence of marA or at least in reduced production) (George et al, 1995) and susceptible AG100A/ pET28a-AcrB strain with acrB (plasmid containing the gene encoding AcrB leading to AcrAB-TolC overexpression as in [7/ 9] (7.7) [1/ 3] (1.7) [2/ 5] (3.2) [545/727] (618.4) [0/ 2] (0.7) [−0.31/ 2.91] (1.04) 5/ 6.4 (5.88) [3/ 5] (3.5) [1/ 3] (1.8) Fluoronaphthyridines Tosufloxacin, gemifloxacin, trovafloxacin, enoxacin GC7368/pCLL3431) (Li et al, 2011;…”

“…For this purpose, we also plotted MIC data from wild-type E. coli strains (Figure 1B). We selected different wild-type strains, such as: AG100 (Cohen et al, 1988;Okusu et al, 1996;Alekshun and Levy, 1997;Oethinger et al, 2000;Randall and Woodward, 2002;Visalli et al, 2003;Nicoloff et al, 2006;Bohnert et al, 2008;Keeney et al, 2008;Wehmeier et al, 2009;Yilmaz et al, 2015;Schuster et al, 2016;Song et al, 2016;Vergalli et al, 2020;Ferrand et al, 2023), ATCC25922 reference strain from EUCAST 2023 (Chollet et al, 2004;Aires and Nikaido, 2005;Lim Nikaido, 2010;Li et al, 2011;Sutcliffe et al, 2013;Whalen et al, 2015; Société Française de Microbiologie, 2023), JM101 (George et al, 1995), BW1556 (Munro and Kell, 2022) and BW25113 (Plé et al, 2022). Not all antibiotics have been assessed against strains overproducing AcrAB-TolC.…”